The present invention relates generally to the binding, transport and infection of cells by retroviruses including HIV-1, and HIV-2. Methods of identifying agents that modulate such processes, the agents themselves and therapeutic uses of such agents are also provided. Related diagnostic methods are also included.
The human immunodeficiency viruses infect CD430  macrophages and T helper cells. Although HIV-1 entry requires cell surface expression of CD4, to which the viral envelope glycoproteins bind, several studies have suggested that it is not sufficient for fusion of the viral envelope to the cellular plasma membrane. Early studies have shown that while human cells expressing a transfected CD4 gene were permissive for virus entry, murine cells expressing human CD4 were not. These findings led to the suggestion that there is a species-specific cell surface cofactor required in addition to CD4 for HIV-1 entry. Subsequent studies have shown that strains of HIV-1 that had been adapted for growth in transformed T-cell lines (T-tropic strains) could not infect primary monocytes or macrophages; in contrast, primary viral strains were found to infect monocytes and macrophages, but not transformed T cell lines. This difference in tropism was found to be a consequence of specific sequence differences in the gp120 subunit of the envelope glycoprotein, suggesting that multiple cell type-specific cofactors may be required for entry in addition to CD4.
The nature of the cofactors required for HIV entry proved elusive until it was recently discovered that the principal receptor for entry of macrophage-tropic (M-tropic) HIV-1 strains was CCR5, whereas the principal receptor for entry of T-cell line-tropic (T-tropic) strains was CXCR4. On the other hand, both M-tropic and T-tropic strains of simian immunodeficiency virus (SIV) can be mediated by CCR5, but not CXCR4 [Chew et al., J. Virol, 71:2705-2714 (1997); Marcon et al., J. Virol, 71:2522-2527 (1997); and Edinger et al., Proc. Natl. Acad. Sci. U.S.A., 94:4005-4010 (1997)]. More importantly, SIV strains were also found to infect CD430  cells that lack CCR5 [Chen et al., J. ViroL., 71:2705-2714 (1997); and Edinger et al, Proc. Natl. Acad. Sci. U.S.A., 94:4005-4010 (1997)].
In humans, CCR5-tropic viruses are primarily involved in transmission, while viruses with broader tropism, particularly for CXCR4, emerge during progression to immunodeficiency [Fauci, Nature, 384:529-534 (1996)]. It is not yet known whether appearance of CXCR4-tropic viruses is a consequence or the cause of immune system decline. Insight into this key problem of virus evolution is likely to require experimental manipulation in animal models. Infection of non-human primates with SIV is remains the only good animal model for studying pathogenesis of the immunodeficiency viruses [Desrosiers, Annu Rev Immunol, 8:557-578 (1990)]. Moreover, different species of non-human primates vary widely in their responses to SIV infection. For example, Rhesus macaques succumb to immunodeficiency that closely resembles AIDS in humans, but sooty mangabeys and African green monkeys can sustain infection with little evidence of immune system damage [Kestler, Science, 248:1109-1112 (1990)]. These interspecies differences provide important clues for understanding and combating disease progression in HIV-infected humans.
Transmission of Human Immunodeficiency Virus Type 1 (HIV-1) infection in humans requires the dissemination of virus from sites of infection at mucosal surfaces to T cell zones in secondary lymphoid organs, where extensive viral replication occurs in CD430  T-helper cells and macrophages [Fauci, Nature, 384(6609):529-534 (1996)]. These cells express both CD4 and the chemokine receptor CCR5, which together form the receptor complex required for entry by the R5 viral isolates that are prevalent early after infection [Littman, Cell, 93:677-680 (1998); Lu et al., Proc. Natl. Acad. Sci. U.S.A., 94(12):6426-6431 (1997); Dragic et al, Nature, 381:667-673 (1996); U.S. Pat. No: 5,939,320, Issued Aug. 17, 1999; and U.S. patent application 09/116,498, Filed Jul. 7, 1998, the contents of which are hereby incorporated by reference in their entireties]. Viruses with tropism for other chemokine receptors, particularly CXCR4, are rarely transmitted, and generally appear only late in infection. Such CXCR4-tropic isolates replicate poorly in macrophages, and it has hence been proposed that infection of macrophages is a requisite component of viral transmission.
The mechanism of early viral dissemination remains vague, but based on anatomical distribution of different hematopoietic lineage cells and on in vitro infectivity studies it has been inferred that immature dendritic cells (DC) residing in the skin and at mucosal surfaces are the first cells targeted by HIV-1. DC are the most potent antigen-presenting cells in vivo [Banchereau and Steinman, Nature, 392:245-252 (1998); Valitutti et al., Nature, 375:148-151 (1995)]. Immature DC in peripheral tissues capture antigens efficiently and have a unique capacity to subsequently migrate to the T cell areas of secondary lymphoid organs. As the cells travel, they mature and alter their profile of expression of cell surface molecules, including chemokine receptors, lose their ability to take up antigen, and acquire competence to attract and activate resting T cells in the lymph nodes [Banchereau and Steinman, Nature, 392:245-252 (1998); Adema et al., Nature, 387(6634):713-717 (1997)]. HIV-1 is thought to subvert the trafficking capacity of DC to gain access to the CD4+to T cell compartment in the lymphoid tissues [Steinman and Inaba, J. Leukoc. Biol., 66(2):205-208 (1999); Rowland-Jones, S. L., Curr. Biol., 9(7):R248-R250 (1999); and Grouard and Clark, Curr. Opin. Immunol., 9(4):563-567 (1997)].
Immature DC express CD4 and CCR5, albeit at levels that are considerably lower than on T cells [Granelli-Pipemo et al., J. Exp. Med., 184:2433-2438 (1996); Rubbert et al, J. Immunol., 160(8):3933-3941 (1998)], and they have been reported to be injectable with R5 strains of HIV-1. In contrast, immature DC do not express CXCR4 and are resistant to infection with CXCR4-tropic isolates of HIV-1 [Granelli-Pipemo et al., J. Virol., 72:2733-2737 (1998); Blauvelt et al., J. Clin. Invest., 100:2043-2053 (1997); and Weissman et al., Proc. Natl. Acad. Sci. U.S.A., 92:826-830 (1995)]. Entry of HIV-1 into immature DC has also been reported to proceed through a CD4-independent mechanism [Blauvelt et al., J. Clin. Invest., 100:2043-2053 (1997)], suggesting that receptors other than CD4 could be involved. There have been conflicting reports regarding the significance of HIV-1 replication within DC [Canque et al., Blood, 93(11):3866-3875 (1999); Ayehunie et al., Blood, 90(4):1379-1386 (1997); Cameron et al., J. Leukoc. Biol., 56(3):257-265 (1994)]. Although replication can be observed in some circumstances, it has also been reported that, in immature DC, replication is incomplete and that only early HIV-1 genes are transcribed.
It has been proposed that virus-infected immature DC migrate to the draining lymph nodes where they initiate both a primary anti-viral immune response and a vigorous productive infection of T cells, allowing systemic distribution of HIV-1 [Cameron et al., Science, 257(5068):383-387 (1992); Weisman et al., Proc. Natl. Acad. Sci. U.S.A., 92:826-830 (1995)]. However, in a non-human primate model of mucosal infection with the simian immunodeficiency virus, it has been difficult to demonstrate productive infection of DC despite rapid dissemination of virus [Stahl-Henning et al., Science, 285(5431):1261-1265 (1999)]. Other efforts to model primary HIV-1 infection in vitro by exposing DC derived from skin or blood to HIV-1 have indicated that these cells are poorly infected. Nevertheless, only DC, and not other leukocytes including monocytes, macrophages, B cells and T cells were able to induce high levels of infection upon co-culture with mitogen-activated CD430  T cells after being pulsed with HIV-1 [Cameron et al., Science, 257(5068):383-387 (1992); Granelli-Pipemo et al., Curr. Biol., 9:21-29 (1999); Weissman et al., Proc. Natl. Acad. Sci. U.S.A., 92:826-830 (1995); Blauvelt et al., J. Clin. Invest., 100:2043-2053 (1997); Cameron et al., J. Leukoc. Biol., 59(2):158-171 (1996)]. In an early study, Cameron et al. [Science, 257(5068):383-387 (1992)] proposed that DC have a unique ability to xe2x80x9ccatalyzexe2x80x9d infection of T cells with HIV, but do not become infected themselves.
Therefore, there is a need to identify the protein or proteins involved in the mediation of HIV from the mucosal surfaces to the T cell areas of secondary lymphoid organs.
Further, there is a need to design methods of identifying agents that will interfere with this mediation.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides the missing link between viral breach of host mucosal defense and infection of T cells in the lymphatic organs by demonstrating that a membrane-bound receptor that is specifically expressed on dendritic cells, DC-SIGN, and which has the amino acid sequence of SEQ ID NO:2, facilitates infection of T lymphocytes by HIV. The present invention further provides new intervention strategies and novel approaches towards prevention, preventive vaccination and therapy against HIV infection based, at least in part, on this finding.
The present invention therefore provides an antibody that is specific for an antigenic fragment of gp120. In one embodiment the antigenic fragment is obtained from a portion of gp120 that binds to DC-SIGN. In another embodiment the antigenic fragment is obtained from the portion of gp120 that is exposed upon gp120 binding of DC-SIGN due to the concomitant conformational change that occurs upon DC-SIGN binding gp120. The present invention also provides antibodies to the portion of DC-SIGN that interacts with T cells and/or macrophages. In a preferred embodiment the antibody is specific for the DC-SIGN-gp120 binding complex. The antibodies of the present invention preferably interfere with dendritic cells facilitating the trans-enhancement of HIV entry into T cells or macrophages.
In a particular embodiment the antibody is a polyclonal antibody. In another embodiment the antibody is a monoclonal antibody. In still another embodiment the antibody is a chimeric antibody. In one such embodiment the antibody is a humanized antibody. The present invention also provides immortal cell lines that produce the monoclonal antibodies of the present invention.
The present invention also provides vaccines and immunogenic compositions. Such vaccines and immunogenic compositions preferably comprise adjuvants. One such immunogenic composition comprises an adjuvant and an antigenic fragment obtained from a portion of gp120 that binds to DC-SIGN. In another embodiment the immunogenic composition comprises an adjuvant and an antigenic fragment obtained from the portion of gp120 that is exposed upon gp120 binding of DC-SIGN due to the concomitant conformational change that occurs upon DC-SIGN binding gp120. In yet another embodiment the immunogenic composition comprises an adjuvant and an antigenic fragment of DC-SIGN that interacts with T cells and/or macrophages. In still another embodiment the immunogenic composition comprises an adjuvant and the DC-SIGN-gp120 binding complex or fragment thereof.
The present invention further provides a soluble form of human DC-SIGN. The soluble form of human DC-SIGN can be used in drag assays or in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
In addition, the present invention provides a mammalian cell that is transfected with a vector encoding human DC-SIGN. Preferably this cell can facilitate the trans-enhancement of HIV entry into a T cell or macrophage. More preferably, the cell lacks the translocation promoting agent and/or CD4 required for HIV entry. In a particular embodiment the mammalian cell is not a dendritic cell. In a preferred embodiment the mammalian cell is a human cell. In particular embodiment, the cell is attached to a solid support matrix.
The present invention further provides methods of filtering a biological fluid to remove a virus expressing an HIV envelope glycoprotein that binds DC-SIGN. In one embodiment the method comprises passing the biological fluid through a solid support comprising DC-SIGN. A related embodiment comprises passing the biological fluid through a solid support comprising a mammalian cell that is transfected with a vector encoding human DC-SIGN. In another embodiment the biological fluid is contacted with a mammalian cell that is transfected with a vector encoding human DC-SIGN and then the cell is removed. In still another embodiment the biological fluid is contacted with a dendritic cell and then the dendritic cell is removed.
The present invention further provides transgenic non-human mammals. In one embodiment the transgenic non-human mammal comprises DNA constructs encoding CD4, one or more translocation promoting agents, and DC-SIGN. Preferably the CD4, the translocation promoting agent(s), and the DC-SIGN are human proteins. In a preferred embodiment, the transgenic non-human mammal is capable of/susceptible to mucosal uptake of HIV, transport of the HIV to lymphatic organs and infection of a target T cell and/or macrophage by HIV. More preferably the infection with HIV (or suitable vector analog) is greatly enhanced relative to a transgenic non-human mammal that does not contain a DNA construct encoding DC-SIGN. In a preferred embodiment the transgenic non-human mammal further comprises a DNA construct encoding cyclin T (which is preferably human cyclin T).
In one embodiment the human translocation promoting agent is CCR3. In another embodiment the hum an translocation promoting agent is CXCR4. In yet another embodiment the human translocation promoting agent is CCR4b. In still another embodiment the human translocation promoting agent is Bob. In yet another embodiment the human translocation promoting agent is Bonzo. In a preferred embodiment the human translocation promoting agent is CCR5.
The transgenic non-human mammal can be any mammal including a primate or rodent including rabbits, rats or monkeys and chimpanzees. In a preferred embodiment the transgenic non-human mammal is a mouse.
The present invention further provides methods of treating or preventing HIV infection by inhibiting dendritic cells from facilitating the trans-enhancement of HIV entry into a cell. One such embodiment comprises administering a humanized form of an antibody of the present invention to the subject. Another embodiment comprises administering a soluble form of DC-SIGN of the present invention to the subject. Yet another embodiment comprises administering a compound identified through an assay of the present invention to the subject. In a preferred embodiment, the cell for which the trans-enhancement of HIV entry is inhibited is a T cell or macrophage.
The present invention further provides methods of identifying a compound that interferes with the trans-enhancement of HIV entry into a cell. In one such embodiment, the compound interferes with viral capture. One such embodiment comprises contacting a first cell with a vector in the presence of a test compound. The vector comprises a viral envelope protein that binds DC-SIGN, whereas the first cell expresses DC-SIGN. A DC-SIGN-viral envelope protein complex forms between the first cell and the vector in the absence of the test compound and the effect of the test compound on the formation of the complex is thus being tested. The unbound vector is separated from the first cell (preferably so is the test compound) and the first cell is next contacted with a second cell. The second cell is susceptible to entry of vectors comprising the viral envelope protein. The amount of vector that has entered the second cell is determined. A test compound is identified as a compound that interferes with the trans-enhancement of HIV entry into a cell when the amount of vector entry determined is less for the case when the test compound was present during the incubation with the first cell and the vector, than when the test compound was absent.
In a particular embodiment removal of the unbound vector (and the test compound) from first cell comprises washing the unbound vector (and the test compound) away from the first cell. In a particular embodiment the second cell is a T cell or macrophage. In another embodiment the second cell expresses human CD4 and a human translocation promoting agent. In one embodiment the human translocation promoting agent is CCR3. In another embodiment the human translocation promoting agent is CXCR4. In yet another embodiment the human translocation promoting agent is CCR2b. In still another embodiment the human translocation promoting agent is Bob. In yet another embodiment the human translocation promoting agent is Bonzo. In a preferred embodiment the human translocation promoting agent is CCR5.
In a particular embodiment the human translocation promoting agent is CCR5 and the viral envelope glycoprotein is JRFL. In another embodiment the vector contains a marker protein. In one such embodiment the amount of vector that has entered the second cell is determined by detecting the amount of marker protein expressed in the cell. In a particular embodiment the marker protein is luciferase. In another embodiment the marker protein is green fluorescent protein. The present invention further provides the compounds identified by these methods.
The present invention also provides an assay for identifying an agent for use in the treatment or prevention of HIV infection using a transgenic non-human mammal of the present invention. One such method comprises administering a test compound to the transgenic non-human mammal and infecting the transgenic non-human mammal with a virus or viral vector having an HIV envelope glycoprotein. The ability of the transgenic non-human mammal to resist the infection is then determined or measured. A test compound is identified as an agent for use in the treatment or prevention of HIV infection when the measured ability of the transgenic mammal to resist the infection is statistically greater in the presence of the test compound than in the absence of the test compound. Again the compounds identified by this method are also part of the present invention.
The present invention further provides a method of identifying a compound that interferes with the trans-enhancement of HIV entry into a cell. One such embodiment comprises contacting a first cell, a vector and a second cell in the presence of a test compound. The vector comprises a viral envelope protein that binds DC-SIGN, the first cell expresses DC-SIGN, and a DC-SIGN-viral envelope protein complex forms between the first cell and the vector in the absence of the test compound. In addition, the second cell is susceptible to entry of vectors comprising the viral envelope protein. The amount of vector that has entered the second cell is then determined. A test compound is identified as a compound that interferes with the trans-enhancement of HIV entry into a cell when the amount of vector entry is less in the presence of the test compound than in its absence. In a particular embodiment the second cell is a T cell or macrophage. In another embodiment the second cell expresses human CD4 and a human translocation promoting agent. In one embodiment the human translocation promoting agent is CCR3. In another embodiment the human translocation promoting agent is CXCR4. In yet another embodiment the human translocation promoting agent is CCR2b. In still another embodiment the human translocation promoting agent is Bob. In yet another embodiment the human translocation promoting agent is Bonzo. In a preferred embodiment the human translocation promoting agent is CCR5.
In a particular embodiment the human translocation promoting agent is CCR5 and the viral envelope glycoprotein is JRFL. In another embodiment the vector contains a marker protein. In one such embodiment the amount of vector that has entered the second cell is determined by detecting the amount of marker protein expressed in the cell. In a particular embodiment the marker protein is luciferase. In another embodiment the marker protein is green fluorescent protein. The present invention further provides the compounds identified by these methods. Accordingly, it is a principal object of the present invention to provide compounds that can be used in the treatment of AIDS.
It is a further object of the present invention to provide assays for identifying new compounds in the treatment of HIV infection.
It is a further object of the present invention to provide soluble fragments of DC-SIGN.
It is a further object of the present invention to generate a soluble form of DC-SIGN that inhibits trans-enhancement of HIV entry into T cells that is facilitated by DC-SIGN.
It is a further object of the present invention to identify DC-SIGN polymorphisms that do enable dendritic cells to facilitate trans-enhancement of HIV entry in T cells.
It is a further object of the present invention to provide methods of identifying drugs that can treat HIV infection.
It is a further object of the present invention to provide methods for making a transgenic animal model for AIDS.
It is a further object of the present invention to provide a vaccine to prevent or retard the onset of AIDS.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.